Conventionally, antigen-specific antibody-producing hybridomas have been prepared to produce monoclonal antibodies. In the conventional method of preparing hybridomas, hybridomas are prepared, after which hybridoma clones producing antigen-specific antibodies are screened. However, the preparation of hybridomas is not efficient. That is, not all B lymphocytes become hybridomas; only some of the B lymphocytes in which cell fusion with a myeloma has occurred become hybridomas. Even when a hybridoma is produced with a spleen cell that has been stimulated with an antigen, not just antigen-specific antibody-producing hybridomas are produced; most of the hybridomas that are produced either produce unrelated antibodies or do not produce antibodies at all.
For example, when looking for a hybridoma producing a target antibody by the conventional method, spleen cells taken from an immunized mouse are subjected to cellular fusion with myelomas and sown into about ten 96-well plates. More could be sown if all the cells were used, but there are time limits when a single person is doing the screening, and those that remain are stored by freezing or the like. Normally, hybridomas grow in about 500 wells when using this method.
The hybridomas in the 500 wells do not all proliferate at the same speed; some grow quickly and others grow slowly. Accordingly, it is impossible to check the growth of all 500 simultaneously. First, a check must be made under a microscope as to which wells have produced cell growth, and whether the number of cells has increased sufficiently to check for antibodies. Subsequently, cell supernatant is collected from suitable wells, and a check is made for the production of antigen-specific antibodies. It is necessary to perform the cell check and cell supernatant check extremely rapidly. This is because hybridomas grow steadily and proliferate excessively if left alone, depleting the nutrients in the medium and dying out. Accordingly, screening must be completed before the desired hybridomas die.
Further, once a well is found in which a target hybridoma is growing, frequently there will be hybridomas producing other antibodies growing in the well in addition to the hybridoma producing the target antibody. Further, since hybridomas drop their own chromosomes while growing, there are also cases where a hybridoma that has been producing an antibody ends up losing the chromosome with the antibody and becomes unable to produce the antibody. The growth of such cells is often more rapid than that of hybridomas that are producing antibody, and most of the cells that are cultured when left alone end up becoming non-antibody-producing cells. Accordingly, when a well is discovered in which a desired hybridoma is growing, the cells in that well are immediately reseeded one cell per well in a 96-well plate (critical dilution method), and screening is conducted again for desired antibody-producing hybridomas (secondary screening). Once a targeted hybridoma has been detected, it is necessary to rapidly proceed through secondary screening before the state of the cell deteriorates.
As set forth above, since screening is sometimes conducted with just some of the hybridomas that are prepared, without screening them all, it becomes difficult to obtain low-frequency antigen-specific antibody-producing hybridomas.
More specifically, in the case of human antigen-specific antibodies, there exists a method of screening cells producing antigen-specific antibodies in strains developed by transforming peripheral B lymphocytes with EB virus (Non-patent Document 1). In this method, since the frequency of the lymphocyte cell strains established is low, the probability of obtaining an antigen-specific antibody-producing B lymphocyte cell strain is extremely low. Further, it takes about a month to establish a cell strain. Still further, the B lymphocyte strains that are established produce only small quantities of antibody. Although hybridomas can be prepared for mice, no system for producing hybridomas with good efficiency has been developed for humans.
Hybridomas can be prepared for mice. Conventionally, to produce a hybridoma, a mouse is immunized with an antigen, the spleen or lymph nodes of the mouse are removed, lymphocytes are prepared, about 108 of the lymphocytes prepared and about 107 myeloma cells are fused using polyethylene glycol or by subjecting them to a voltage, they are cultured in a selection medium such as HAT, the hybridomas that grow are screened by ELISA, flow cytometry, or the like to determine whether or not they are producing the antigen-specific antibody, and the antigen-specific antibody-producing hybridomas are selected (Non-patent Documents 2 and 3). When employing this method, hybridomas grow in 300 to 400 wells. Of these, hybridomas producing antigen-specific antibodies grow in only several percent of the wells. This number varies with the antigen employed, but, it is difficult to prepare hybridomas by this method when the frequency of the antigen-specific antibody-producing B lymphocyte is low.
Accordingly, the present inventors examined methods of conveniently selecting lymphocytes reacting specifically with prescribed antigens in the form of both antigen-specific lymphocytes of relatively high frequency and antigen-specific lymphocytes of low frequency, and preparing antigen-specific antibody-producing hybridomas from the antigen-specific B lymphocytes that were selected. They then devised a method for preparing antigen-specific antibody-producing hybridomas by culturing selected antigen-specific B lymphocytes and fusing the antigen-specific B lymphocytes grown by culturing with myeloma cells to prepare hybridomas, and applied for a patent (Patent Document 1).
Attempts have also been made to specify and select individual cells, and use the cells that have been selected. For example, the separate detection of individual antigen specificity, the recovery of a single detected antigen-specific lymphocyte, and the use of the single antigen-specific lymphocyte recovered to prepare an antibody, for example, have been examined (Patent Documents 2 and 3).    [Patent Document 1] WO2004/087911    [Patent Document 2] Japanese Patent Un-examined Publication 2004-173681    [Patent Document 3] Japanese Patent Un-examined Publication 2004-187676    [Non-patent Document 1] “Methods of Detecting Lymphocyte Functions (Version 5)”, Junichi YANO, Michio FUJIWARA, eds., Chugai Igakusha (1994), “Use of EB virus transform B cells for preparation of human monoclonal antibody”, Fumio MIZUNO, Toshiro OHSATO, pp 381-391.    [Non-patent Document 2] “Methods of Detecting Lymphocyte Functions (Version 5)”, Junichi YANO, Michio FUJIWARA, eds., Chugai Igakusha (1994), “Preparation of monoclonal antibody with B cell hybridomas”, Hideo NARIUCHI, pp 574-576    [Non-patent Document 3] Monoclonal antibodies in “Antibodies: A Laboratory Manual” by Ed Harlow and David Lane, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp 139-pp 244, 1988    [Non-patent Document 4] In vitro antibody production. In Current Protocols in Immunology. Edited by J. E. Coligan et al., John Wiley & Sons (New York), p. 3.8.1-3.8.16, 1991.    [Non-patent Document 5] Babcook, J. S., Leslie, K. B., Olsen, O. A., Salmon, R. A., Schrader, J. W. A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Proc Natl Acad Sci USA, 93: 7843-7848, 1996.    [Non-patent Document 6] Measurement of polyclonal immunoglobulin synthesis using the ELISPOT assay. In Current Protocols in Immunology. Edited by J. E. Coligan et al., John Wiley & Sons (New York), p. 7.14.1-7.14.7, 1991.    [Non-patent Document 7] Assays for antibody production. In Current Protocols in Immunology. Edited by J. E. Coligan et al., John Wiley & Sons (New York), p. 2.1.1-2.1.22, 1991.    [Non-patent Document 8] J. C. Love, J. L. Ronan, G. M. Grotenbreg, A. G. van der Veen, H. L. Ploegh, A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nature Biotechnology, 24: 703-707, 2006.
The entire contents of Patent Documents 1-3 and Non-patent Documents 1-8 are hereby incorporated by reference.
In the above-described conventional methods, the separate detection of the antigen specificity of individual lymphocytes and the recovery of the antigen-specific lymphocytes that are detected are conducted manually. In Patent Documents 2 and 3, it is stated that the specification of cells at the cellular level has been confirmed, and that it is also possible to recover the specified cells. However, the actual detection of those lymphocytes that have specifically reacted with an antigen from among numerous lymphocytes is difficult.
As an example of a detection method, the fact that the calcium ion concentration rises in lymphocytes that have reacted with an antigen is exploited, this change in calcium ion concentration is detected by fluorescence, and antigen-specific lymphocytes are specified. However, depending on the type of cell (lymphocyte), the method of generating fluorescence, the intensity thereof, and the like may differ. There are lymphocytes in which the calcium ion concentration rises immediately upon antigen stimulation, resulting in an increase in the intensity of fluorescence. However, there are also cells in which the calcium ion concentration rises only after a certain period has elapsed following antigen stimulation, resulting in a rise in the intensity of fluorescence. Further, it is necessary to measure nearly simultaneously and in one shot the intensity of fluorescence of about 10,000 to 200,000 lymphocytes being held on the surface of a several centimeter square tube. Still further, since this fluorescence arises in individual cells, the intensity of the fluorescence is low, requiring highly sensitive fluorescence detection.
Thus far, there exists neither a method nor a device capable of simultaneously measuring the intensity of numerous sources of weak fluorescence gathered in highly concentrated fashion on the surface of a tube.
One conventional device is the laser scanning cytometer. With a laser scanning cytometer, it is possible to measure the concentration of calcium in several hundred thousand individual cells. However, a single scan requires a lengthy 10 minutes or more, precluding real-time detection in lymphocytes the fluorescence intensity of which changes several minutes after antigen stimulation.
A fluorescent microscope with projector is comprised of an ordinary fluorescent microscope combined with a projection device. A fluorescent microscope with projector presents no problem in terms of speed. However, as an ordinary microscope, the scope of projection is narrow, making it possible to detect fluorescence in only about 1,000 cells at a time. It is impossible to detect fluorescence in several tens of thousands to several hundred thousand cells at a time.
A cell chip detector based on a DNA microarray scanner is a device that has been developed to detect fluorescence in cells arrayed in cell chips, and can detect fluorescence inside or outside cells. However, it is a system that excites a fluorescent dye with a laser and detects the excitation light. Thus, in detection systems that track changes over time, the line scanning rate of the laser is slow, precluding the simultaneous detection of numerous cell regions.
In the methods described in above-cited Patent Documents 2 and 3, the reaction detection system for detecting cells reacting specifically to an antigen is required to have the ability to capture in real time changes in calcium over time within individual cells and the ability to detect individual cells that are present in only small number (react only slightly) among large numbers of cells, necessitating the ability to separately and simultaneously analyze large numbers of cells in parallel.
Accordingly the object of the present invention is to provide a method permitting the simultaneous measurement of the states of a large number of cells, exceeding 10,000 and desirably exceeding 100,000, being held on a chip, such as the reactive properties of antigen-stimulated lymphocytes, and the separate determination of the states of individual cells.
B lymphocytes express a single antibody on the cell surface. When a pathogen (antigen) or the like invades the body, the antigen binds to the antibody on the cell surface, activating the cell and causing it to proliferate and differentiate. Finally, it differentiates into an antibody-secreting cell. There exist already numerous methods of specifying antibody-secreting cells. Typical methods of specifying single antibody-secreting cells are the plaque method and the Enzyme-Linked Immunospot (ELISPOT) method.
The plaque method is a method of specifying antigen-specific antibody-secreting cells in which hemolysis induced by binding antibodies to erythrocytes that have been labeled with the antigens surrounding antibody-secreting cells is employed as an indicator (Non-patent Document 4). A method that detects antigen-specific antibody-secreting cells by the plaque method and recovers the antibody gene has already been developed (Non-patent Document 5).
The ELISPOT method is a method of inoculating cells on a plate that has been coated with antigen, causing the antibody that is secreted by antibody-secreting cells to bind to the antigen around the cells, and detecting this with enzyme-labeled anti-Ig antibody or the like (Non-patent Document 6). With the ELISPOT method, in the course of detecting the binding of antigen-specific antibody around cells with enzyme-labeled anti-Ig antibody, the cells themselves get washed away, precluding recovery of the antigen-specific antibody-secreting cells.
Hybridomas are antibody-secreting cells. However, in the course of screening hybridomas, enzyme-linked immunosorbent assay (ELISA) is commonly employed (Non-patent Document 7). Recently, this method has been further developed; a method has been reported whereby individual hybridomas are cultured in small wells, and the antibody that is secreted in each well is detected by an antigen or the like labeled with a fluorescent label to detect antigen-specific antibody-secreting cells (Non-patent Document 8).
To achieve the above-stated object of the present invention, the present inventors conducted extensive research into providing a method for detecting immune cells, such as new antigen-specific antibody-secreting cells, that was completely different from the methods described in Non-patent Documents 4 to 8. The present invention was devised on that basis.